Para-hydroxycinnamic acid (pHCA) is a high-value, aromatic chemical compound that may be used as a monomer for the production of Liquid Crystal Polymers (LCP). LCPs are used in liquid crystal displays, and in high speed connectors and flexible circuits for electronic, telecommunication, and aerospace applications. Because of their resistance to sterilizing radiation and their high oxygen and water vapor barrier properties, LCPs are used in medical devices, and in chemical and food packaging. Due to its importance as a high value, aromatic chemical compound, chemical synthesis of pHCA is known. However, these chemical methods are expensive due to the high cost of the starting materials and the extensive product purification required. Moreover, these methods generate large amounts of unwanted byproducts.
Biological production of pHCA offers an alternative to chemical synthesis of this material. In plants, pHCA (also known as p-coumarate) is made as an intermediate for the synthesis of various secondary metabolites such as lignin [Plant Biochemistry, Ed. P. M. Dey, Academic Press, (1997)] and isoflavonoids. Phenylalanine ammonia-lyase (PAL) converts L-phenylalanine to trans-cinnamic acid (CA), which is then converted to pHCA. Methods of pHCA isolation and purification from plants are known [R. Benrief, et al., Phytochemistry, 47, 825-832; (1998)], however, these methods are time consuming and cumbersome and do not therefore provide an economical alternative to the current chemical synthesis route. PAL enzymes are also found in fungi (Bandoni et al., Phytochemistry 7:205-207 (1968)), yeast (Ogata et al., Agric. Biol. Chem. 31:200-206 (1967)), and Streptomyces (Emes et al., Can. J. Microbiology 48:613-622 (1970)), but not in Escherichia coli mammalian cells (Hanson and Havir In The Enzymes, 3rd ed.; Boyer, P., Ed.; Academic: New York, 1967; pp 75-167).
Some PAL enzymes, in addition to their ability to convert phenylalanine to cinnamate, can accept tyrosine as a substrate. The tyrosine ammonia lyase (TAL) activity of these enzymes directly converts tyrosine to pHCA. Generally, these enzymes have much higher PAL than TAL activities. A few enzymes with higher TAL than PAL activities have been found, including PAL/TAL enzymes from the bacterium Rhodobacter capsulatus (Kyndt et al., FEBS Letters 512:240-244 (2002)), the yeast, Rhodotorula glutinis (also known as Rhodosporidium glutinis and Thodosporidium toruloides; PAL/TAL58; Hanson and Havir, In The Biochemistry of Plants; Academic: New York, 1981; Vol. 7, pp 577-625), the yeast Trichosporon cutaneum (U.S. Pat. No. 6,951,751), and the bacterium Rhodobacter sphaeroides (US20040059103). In addition, U.S. Pat. No. 6,368,837 discloses a mutagenized Rhodosporidium toruloides PAL/TAL with an increased TAL/PAL activity ratio over that of the wild type enzyme. Several other mutant PAL/TAL genes that encode enzymes with enhanced TAL activity are disclosed in U.S. Pat. No. 6,521,748. Several of these enzymes with high TAL activity have been introduced into microorganisms for production of pHCA (U.S. Pat. No. 6,368,837, US20040059103 A1). These engineered microorganisms expressing TAL activity can be used in fermentation processes for production of pHCA. However, the enzymes having TAL activity used in these biocatalysts are sensitive to high temperatures. It is desirable to have a more robust enzyme with high TAL activity for production of pHCA using engineered biocatalysts. Applicants have solved the stated problem by isolating, characterizing and expressing a TAL enzyme which exhibits high levels of TAL catalytic activity at high temperatures.